This invention relates to water soluble fluors. More specifically, it relates to a new class of water soluble fluors, their preparation and their use as enhancers for radioactivity detection in auto-radiography.
Auto-radiography is the production of an image in a photographic emulsion by a radioactivity labelled substance. Auto-radiography is an important method in biological, biochemical, and clinical investigations and analyses. One of its principal uses is to locate bands of radio labelled materials, e.g., molecules radio labelled with .sup.3 H, .sup.14 C, .sup.32 P, .sup.35 S, or .sup.125 I on chromatography plates or on slab gels used for electrophoresis. The usefulness of auto-radiography, however, has frequently been limited because of the long exposure times usually required for the low radiation levels of isotopes incorporated into the plates and gels and, in some cases, the very weak energies of radiation.
One approach taken to overcome that problem has been the use of scintillation materials which act as amplifiers of the exposure process. Radiation energy causes the fluor present in the system to emit light on a certain wavelength, and the light exposes the photographic emulsion in a way that is much faster and more efficient than could be obtained by relying on auto-radiography alone. The combination of autoradiography and fluorescence is called fluorography or autofluorography. Bands of material labelled with radioactive isotopes can be detected more readily and rapidly in, for example, slabs of electrophoresis gel, by means of fluorography.
In chromatography and electrophoresis, the radioactive material to be measured is absorbed or adsorbed according to conventional techniques on or in an organic or inorganic absorbent or adsorbent layer or column of separation medium or material, e.g. silica gel, alumina, cellulose, polyamide, polyacrylamide, cross-linked dextran, agarose, etc., which is usually supported on a plate, e.g. glass or plastic sheet. This is called a chromatogram or electrophoretogram.
Two of the most common separation media used in electrophoresis are aqueous polyacrylamide and agarose gels.
Gel electrophoresis is a method of separating charged particles, such as proteins, whereby the charged particles move through a gel medium under the influence on an applied electric field, their rate of movement through the lattice formed by the hydrated gel being dependent on charge and molecular size or weight. When the electric field is removed, the particles are present in the gel in discrete bands which can either be sliced up for liquid scintillation counting, or in the case of radionuclides such as tritium which emit lower energy particles, more preferably analyzed by fluorography.
In the case of radioactive labelled animal tissues, e.g. tissue autoradiography, the radioactive material is usually administered to the live animal and becomes selectively absorbed or adsorbed into certain tissues so that the tissue, usually in the form of a thin slice, may be considered as the absorbent or adsorbent layer. In the case of paper chromatography the paper (cellulose) is the adsorbent.
Where the adsorbent material is in the form of a thin layer supported on a plate, it is called thin layer chromatography and a thin layer chromatogram.
In autoradiography, the radioactivity of the material being tested is measured by a film sensitive to radioactivity.
In autofluorography, a fluor or scintillator, which is excited or stimulated by radioactivity to emit light, is applied in close proximity to the radioactive material and the intensity of light emission is measured by a photographic film, which is sensitive to light. Conventionally, the photograph is taken with the radioactive sample sandwiched against the emulsion of the film.
Fluorography has important advantages over conventional autoradiography, the most important of which is a markedly shorter exposure time (typically shortened from two weeks to 16 to 24 hours) with weak radioactive emitters, such as tritium.
However, in spite of this important advantage, presently known autofluorographic techniques have serious disadvantages, particularly in systems where relatively thick layers of absorbent or adsorbent materials are used in the separation process, e.g., polyacrylamide gel electrophoresis which is frequently used in receptor site, nucleic acid, and enzyme research.
One of the problems is in developing a method for placing and maintaining the scintillator fluor in close proximity to the radioactive emitter. If not in close proximity, a portion of the emitted radioactive particles will not reach the scintillation fluor. In the case of thin layer chromatography, the scintillator fluor has been dissolved in a suitable carrier, e.g. benzene or toluene, and then sprayed onto the thin layer separation medium, e.g. a paper strip, containing the radioactive sample. After drying, a piece of film sensitive to the light emission of the scintillator may then be juxtaposed and this sandwich is allowed to stand for a time sufficient to achieve exposure. In such a system, it is difficult to evenly distribute the scintillator fluor, the radioactive material may spread and diffuse, and the small crystals of scintillator fluor tend to be so loosely bound that great care must be exercised in handling the sample.
In addition to the above disadvantages it is sometimes desirable to use thicker layers of adsorbent or absorbent material. Once any appreciable thickness is used, i.e. greater than about 0.1 mm, the technique of spraying no longer places the scintillator fluor in close enough proximity to enough of the radioactive material. This results in a drastic loss in the ability of the scintillator fluor to be excited by the emitted particles and convert them into light.
Accordingly, it is necessary to somehow transport the scintillator fluor into the interior of the separation medium. One method for accomplishing this transportation is by soaking the separation medium of absorbent or adsorbent material in a bath containing the scintillation fluor dissolved in a suitable carrier.
A number of fluorography systems are currently available. One system was described by Bonner et al, Eur. J. Biochem. 46:No. 1: 83-88 (1974), the disclosure of which is hereby incorporated herein by reference.
In that method the radioactively-labelled protein is separated by electrophoresis using an aqueous polyacrylamide gel, followed by soaking the gel in about 20 times its volume of dimethylsulfoxide (DMSO) for 30 minutes, and then immersed a second time for 30 minutes in fresh DMSO to displace all the water from the gel. The next step is to soak the gel in a 20% (w/w) solution of 2,5-diphenyloxazole (PPO) in DMSO to impregnate the gel with scintillator which is then precipitated in the gel by washing with water. The gel is finally dried and exposed to the film. This technique has numerous disadvantages, many of which are reported in the appendix of an article by Laskey and Mills in the Eur. J. Biochem., Vol. 56, pages 335-341 (1975), incorporated herein by reference. Agarose gels containing less than 2% polyacrylamide (plus 0.5% agarose) or agarose alone dissolved in DMSO unless methanol is substituted for the DMSO. Even this substitution is only effective for gels having less than 2% polyacrylamide, since gels having higher concentrations of polyacrylamide shrink severely when contacted with methanol. Even at 30% methanol, shrinkage of higher polyacrylamide concentration gels may take place. Another disadvantage is that the failure to remove all the DMSO may result in adhesion of the film to the gel and artifactual blackening of the film. Another disadvantage is the ability of DMSO to penetrate through the skin of anyone handling it by itself or the gel which has been soaked in it, thereby carrying toxic dissolved material with it through the skin as well as imparting a garlic smell to the person's breath. Another disadvantage is that the gels must be soaked in the DMSO-fluor solution for as much as 3 hours to obtain complete impregnation. A further disadvantage is that high concentrations of PPO, concentrations between 14% and 19% (w/w) being typical, must be used in the impregnation solution. Another disadvantage with DMSO as well as with other conventional carriers is that while PPO is efficient in converting absorbed radiation into photons of light, it is somewhat limited in its ability to absorb the energy emitted by the radioactive emitter. Another disadvantage is that the soakings in DMSO to dehydrate the gel are time-consuming.
One method for increasing the absorption ability of PPO when thin layer chromatography is being employed is described in Bonner and Stedman, Analytical Biochemistry, Vo. 89, pages 247-256, 1978, incorporated herein by reference. Three methods for detection of .sup.3 H and .sup.14 C in silica gel thin layer chromatograms are described in that article. The first method utilizes 2-methylnaphthalene (2MN) which is described as being a scintillation solvent for use in solid systems by analogy to scintillation fluids which many times contain a solvent in addition to the scintillator. As in liquid systems, the solvent molecules collect the energy from the emitted beta radiation and transfer it to PPO molecules, which then emit photons of light. A solvent is a compound which converts the kinetic energy radiated by the radioactive emitter to electronic excitation energy and transfers that energy to the fluor dissolved therein. The first method comprises dipping the dried thin layer plates in a solution of 2MN which has been liquified by heating and which contains 0.4% (w/v) of PPO, until they are soaked and then removing the plates from the solution. When the solution has solidified, the plate is placed against film and exposed. An alternative, if spraying is deemed to be more desirable, is to replace 10% of the 2MN with toluene to make the solution a liquid at room temperature. The second method involves dipping the plates in an ether solution containing between 7% and 30% (w/v) of PPO, drying the plates and then exposing as above, with better sensitivity being seen as the PPO concentration increases. The third method involves dipping the thin layer plates in melted PPO until soaked, removing and then heating until the excess PPO has drained off, and exposing to film as above. While useful in thin layer chromatography, numerous problems exist in attempting to use such systems with other media. One problem is that neither PPO nor 2MN is soluble nor miscible in water to any appreciable extent. Accordingly aqueous polyacrylamide or agarose gels are not impregnated with PPO nor 2MN while in the hydrated state, nor even if dried since the lattice structure collapses upon drying. Secondly, PPO and 2MN are very expensive even if it were possible to use them in such systems. The second method also is not useful with aqueous gels since ether and similar solvents such as alcohols cause drastic shrinkage of such gels. Furthermore, relatively high (7% to 30%) concentrations of expensive PPO in the ether are required for efficient fluorography.
Another fluorographic system has been described in U.S. Pat. No. 4,293,436, issued Oct. 6, 1981 to Dennis L. Fost, the disclosure of which is also incorporated herein by reference. In that method, the aqueous separation medium or other medium to be subjected to fluorography is impregnated with a water-soluble or water-miscible lower alkyl carboxylic acid in which a scintillator fluor has been dissolved or dispersed, followed by precipitation of the fluor within the gel or tissues by aqueous soaking. However, that procedure also suffers from disadvantages. Handling of the fluors, which are generally soluble only in organic solvents, requires impregnation times in subsequent manipulation steps are long as compared with the times of the present invention, e.g., two to three times the periods required with this invention. The fluorescent system may also fade with time, and its enhancement drop within a relatively short period of time. Further, the treatment with highly concentrated carboxylic acid, and the further tratment of extended aqueous soaking, both tend to adversely affect the sharpness of the bands, thus decreasing the accuracy of the procedure. The carboxylic acids may also cause the gel being treated to swell, and this requires the addition of an anti-swelling agent.
Another system, not necessarily prior art to the present invention, is now being marketed by National Diagnostics under the mark AUTOFLUOR. The exact nature of that material is not known, but a sample obtained some months ago appeared to contain a naphthol disulfonic acid. The material was unstable, and thus could not be utilized effectively after relatively short periods of time. A more recent sample seems to be based on sodium salicylate. For the disadvantages of using sodium salicylate, see J. P. Chamberlain, Anal. Biochem. 98:132-5 (1979).
It is an object of this invention to provide a new auto-fluorographic enhancer, containing water soluble fluors which eliminate the problems associated with the impregnation of gels, and permit wider and more convenient use of fluorography.
Ott et al, J. Am. Chem. Soc., 78:1941 (1942), the disclosure of which is hereby incorporated herein by reference, reported the preparation of 2,5-diphenyl-3-methyloxazolium salts, which were apparently soluble to some degree in water and had some fluor properties. However, these compounds are only stable in acidic solutions, and are rapidly and quantitatively converted to the N-methyl-alpha-acylamido ketone by hydrolytic ring cleavage in alkali. Accordingly, their use as auto-radiographic enhancers is severely limited.
Bodendorf et al, Archiv. der Pharm., 298:293 (1965), the disclosure of which is also incorporated herein by reference, reported the preparation of 4-[(2,5-diphenyloxazolyl)methyl]piperidinium hydrochloride and 4-[(2,5-diphenyloxozolyl)methyl]morpholine. However, those materials were neither synthesized, formulated nor tested for use in fluorography.
Certain water soluble compounds, such as alpha-naphthol polyethylene glycol (Naftaxol-Hoechst), p-octylphenolpolyethylene glycol (Triton-X-100, Rohm & Has) and p-nonylphenylpolyethylene glycol (Igepal CO730, GAF) are known as surfactants. Although those compounds do in fact possess some fluorescent properties, they have not been utilized as enhancers for use in auto-radiography. Typically such materials have low quantum efficiencies, e.g., below 0.2 to well below 0.1, and many such materials may fluoresce at wavelengths which are not suitable for fluorography.